Purpose

Run pilot study on cell growth with concentrations of AgNO3 in solution varying from 1uM to 10mM

Procedure

DH5α-T1 cell pellets were taken out from -80°C freezer and left in room temperature to defrost.

Under a sterile environment, 3mL of autoclaved LB medium were added onto pellet. The solution was pipetted up and down gently to mix.

In five bottles of 125mL Erlenmeyer flasks containing 50mL LB medium, the following amount of LB were taken out of the medium from each flask:

4.76uL, 5.00uL, 50.0uL, 500uL, 5000uL

Respectively, the following amount and concentrations of AgNO3 were placed in:

Volume of LB taken out(uL)

Volume of AgNO3 added(uL)

Concentration of AgNO3 added(M)

Final Concentration of AgNO3 in flask(uM)

4.76

4.76

0.0105

1

5

5

0.1

10

50

50

0.1

100

500

500

0.1

1,000

5000

5000

0.1

10,000

Then, 1mL of resuspended cells were placed into each of the five flasks.

The positive control contains 50mL of LB media and 1mL of cells

One more control was set up containing 45mL of LB media and 5mL of 0.1M AgNO3.

All seven flasks were placed into orbital shaker at 230rpm, at 37°C, for 15 hour time period.

Absorbance on a dual-beam spectrometer was set up under photometric method, wavelength at 600nm, to measure the absorbance of each cell solution at each hour.

3mL of LB solution was used as blank for all measurements, and 3mL of sample was taken each time for absorbance readings.

Results

Seven samples were run for a period of 15 hours, and absorbance at 600nm was taken at every hour.

The absorbance for each sample at each hours were shown in the following table:

Observation

After the addition of 0.1M AgNO3 into 50mL LB medium, the solution immediately turned opaque. Due to the small amount of AgNO3 added into solutions containing AgNO3 with the following concentration: 1uM, 10uM, 100uM, 1mM, and due to the limit of LB medium present, no controls were set for these samples.

The solution containing cells and 10mM AgNO3 appeared to be the most opaque, a control that contained 45mL LB and 5mL 0.1M AgNO3 were also run at the same time.

The opaque color of solution containing cells 10mM AgNO3, as well as the control containing 45mL LB and 5mL of 0.1M AgNO3, turned darker over time, eventually, at the point of 15 hours, the color of solution turned darkish gray.

The opaque color might be due to silver ions in solution being oxidized by the chloride present in LB media, turning a darker color.